In the fields of clinical diagnostics and industrial microbiological control, food-processing, pharmaceuticals or cosmetics, gelled culture media in petri dishes, most frequently agar media, have been an indispensable tool in the detection and identification of pathogenic microorganisms since the end of the 19th century.
Several products have been made commercially available to replace a petri dish culture medium. One of these, the Petrifilm™ system, comprising rehydratable nutrients, is very widely used. Another system developed by Nissui Pharmaceutical, Compact Dry™, also consists of a dehydrated medium. These culture media have the advantage that they can be preserved for longer than a ready-to-use agar culture medium. They may also, as is the case for Petrifilm™, be small in size and thus take up a small amount of incubation space.
Thus, broadly speaking, there are two ways to obtain a rehydratable culture medium: the first consists in placing the culture medium in liquid form in the support, then drying the whole thing, and
the second consists in adhering the culture medium in dehydrated form to a support, so as to immediately obtain a rehydratable culture medium.
The first method, namely obtaining rehydratable nutritive media manufactured including a phase for wet impregnation of the nutrients, has been the subject of several patent applications. Thus, patent application CN102337324 describes a method in which the nutritive broth is mixed with a chemical component which evaporates rapidly. Document WO2005/061013 describes a marker dissolved in a solvent and deposited on an absorbent layer, so as to detect vaginitis. More recently, patent application US20130089887 describes a support, namely a thin membrane impregnated with chromogenic and/or fluorogenic substrates dissolved in a solvent, placed in contact with an agar medium.
Nonetheless, this method for dissolving in water or in a solvent has a negative impact on the length of time for which the rehydratable culture medium can be preserved. Indeed, placing certain fragile products such as enzymes or enzymatic or metabolic substrates or antibiotics in suspension may have a severe impact on their overall stability. The heating required to dry the culture medium may also denature, and render ineffective, the heat-sensitive components of the reaction medium. This method in aqueous phase also does not make it possible to control and vary the location of the reaction medium and/or the various additives required for bacterial visualization.
In order to overcome the drawbacks of the culture media obtained by this method, the second method proposes placing the nutritive powder directly onto the support without a prior phase of dissolving said powder.
Thus, 3M propose a dehydrated nutritive medium coated with adhesive and placed on a film without having gone through a prior phase of dissolving the medium. This device consists of two parts, a bottom film and a top film, covered at their surface by certain components of the dehydrated culture medium. At the time of the analysis, the sample is placed between these two films.
This device and the associated detection method, as described in application WO 2009/082667, have several drawbacks.
First of all, this device requires, for its manufacture, a step of adhering the dehydrated nutrients to the films which have had to be coated with adhesive beforehand. Following this, the culture medium cannot de facto form a three-dimensional structure with variable height and layered concentrations, since it is adhered to a film. Only one small superficial layer of medium is therefore available. The volume of the liquid sample required for microbiological analysis may therefore not exceed 1 or 2 ml, which impacts the threshold for detection sensitivity. Then, the packaging of the Petrifilm™ requires the manufacturing of the bottom film and the top film together. It also does not make it possible to have several different culture media on the same device. Moreover, this device has limited applications and cannot, for example, be used for taking swabs or as a dressing. Finally, the Petrifilm™ necessarily requires the input of an external operator providing the aqueous sample.
On the other hand, in an earlier patent application FR1257047, the applicant proposes a method for isolation, from a sample to be analyzed, on a culture medium that is rehydratable in situ, which makes it possible to obtain isolated colonies. This rehydratable medium is covered with a membrane enabling colonies to be isolated. Thus, the culture medium remains sterile and the colonies develop on the membrane which is just above said medium.
However, isolation of colonies, on an agar or non-agar support, is sometimes seen as a constraint and is often incompatible with experiments carried out outside the laboratory and/or by people having little knowledge and know-how in the field of microbiology.